B-Myb is a highly conserved member of the Myb transcription factor family, which plays an essential role in cell cycle progression by regulating the transcription of genes at the G 2/M-phase boundary. The role of B-Myb in other parts of the cell cycle is less well-understood. By employing siRNA-mediated silencing of B-Myb expression, we found that B-Myb is required for efficient entry into S-phase. Surprisingly, a B-Myb mutant that lacks sequence-specific DNA-binding activity and is unable to activate transcription of B-Myb target genes is able to rescue the S-phase defect observed after B-Myb knockdown. Moreover, we have identified polymerase delta-interacting protein 1 (Pdip1), a BTB domain protein known to bind to the DNA replication and repair factor PCNA as a novel B-Myb interaction partner. We have shown that Pdip1 is able to interact with B-Myb and PCNA simultaneously. In addition, we found that a fraction of endogenous B-Myb can be co-precipitated via PCNA, suggesting that B-Myb might be involved in processes related to DNA replication or repair. Taken together, our work suggests a novel role for B-Myb in S-phase that appears to be independent of its sequence-specific DNA-binding activity and its ability to stimulate the expression of bona fide B-Myb target genes.